The death of ventricular cardiomyocytes is the basis of some of the most severe forms of heart disease. Because the heart has limited repair potential, cell transplantation is being investigated. Given the limitations of both pluripotent stm cells (tumor formation) or fully differentiated cardiomyocytes (poor cell survival, lack of vascula support), an intermediate ventricular progenitor cell (VPC) may be ideal for myocardial repair. But because VPCs are incompletely characterized, no specific cell surface markers are known to enable tracking and isolation of VPCs during development or in vitro differentiation of stem cells. So to pioneer new treatment approaches for heart disease, we propose two specific aims to develop methods to isolate and define VPCs. Aim1: Isolate genetically unmodified mouse ventricular progenitor cells using an antibody targeting Irx4+/SSEA1- ventricular progenitor cells created by domain antibody phage display. We used phage display to pan a library of antibodies against selected VPCs from a line of mouse embryonic stem cells (mESCs) expressing hygromycin resistance under control of Irx4, a gene with an expression profile marking a group of progenitors shown to develop a predominantly ventricular cardiomyocyte fate. An antibody specifically bound VPCs compared to undifferentiated mESCs. In genetically unmodified differentiating mESCs, the antibody specifically recognized a subpopulation of cells that when sorted by FACS was enriched in ventricular-specific genes. Furthermore, immunoprecipitation-mass spectrometry recovered a single dominant protein hit that we will verify is the cognate cell surface receptor of the antibody and assess its role in ventricular cardiogenesis. Finally, we will assess this reagent's ability to purify cells capable of undergoing in vivo differentiation in miceto the cell lineages of ventricular myocardium to benefit a myocardial infarct. Aim2: Isolate human ventricular progenitor cells using nanoflares targeting Irx4 and by creating an induced pluripotent stem cell line expressing mNeonGreen under the control of Irx4 via CRISPR nickase. To isolate human VPCs, we will use spherical nucleic acid probes to label Irx4 mRNA directly in living cells without genetic modification, and CRISPR nickase technology to knock in mNeonGreen in the Irx4 locus of human induced pluripotent stem cells (hiPS). In preliminary experiments, a nanoflare to Irx4 identified Irx4+ cells before the formation of cardiomyocytes in differentiating human stem cells and specifically labeled ventricular over atrial primary cells in mice. 293 cells were successfully targeted by CRISPR nickase technology to knock in mNeonGreen at the Irx4 locus to establish the feasibility of integrating a reporter construct in hiPS cells. We will use these reagents to extend Irx4-based VPC purification to human cells. Impact: The long term goal of this work is to develop methods to isolate mouse and human ventricular progenitor cells during stem cell differentiation without genetic modification so we can characterize the properties of these cells to enable their application for repair of the diseased human heart.